The long-term goal of our studies is to identify the mechanisms mediating the physiological effects of melatonin on human sleep and circadian rhythms, and to apply this knowledge to designing new therapeutic strategies for insomnia. Our recent studies have established that rest state in diurnal genetically well-characterized lower vertebrate, zebrafish, has critical behavioral similarities with sleep and that melatonin can promote a sleep-like state in zebrafish via specific melatonin receptors. The goals of the proposed project are to determine the contribution of overnight melatonin receptor activation by endogenous melatonin to quantitative parameters of nighttime sleep-like state and the circadian rhythm of activity in zebrafish, to identify melatonin receptor subtypes responsible for sleep and circadian modulation, and to identify the reticulospinal neurons mediating locomotor effects of melatonin treatment. The effects of melatonin on sleep-like state and circadian rhythm parameters will be assessed in wild-type and melatonin receptor knockdown larval zebrafish using: i) high throughput automatic image analysis system for locomotor activity recordings; ii) melatonin receptor ligands with different affinity to melatonin receptor subtypes and inhibition of melatonin synthesis; iii) knockdown of melatonin receptor subtypes with antisense morpholino oligonucleotides; iv) in vivo confocal calcium imaging for monitoring spontaneous and evoked activity of individual neurons during high-speed recordings of larval locomotor behavior; v) laser ablation of candidate neurons under confocal microscope, followed by behavioral evaluation. These studies would clarify the role of endogenous melatonin in sleep regulation in the diurnal vertebrate, characterize functional specificity of melatonin receptors and role of reticulospinal neurons in locomotor effects of melatonin. They would also constitute a background for the identification of neuronal networks and intracellular signaling pathways mediating the effects of melatonin on sleep-like state and circadian rhythmicity. Furthermore, a continuation of these studies on the physiological regulation of the sleep-like state in zebrafish, in conjunction with the availability of multiple zebrafish mutants and a complete sequence of zebrafish genome soon to be available, could potentially lead to a better understanding of the genetic basis of sleep regulation and sleep function in vertebrates.